Determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, 2-ethyl-5-methyl-3,3-diphenylpyraline and methadol in meconium by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

This paper details a validated liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) method for the quantification of methadone, and its metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP) and methadol in human meconium. Limits of detection (LOD) were determined to be 1.0 ng/g for methadone, EDDP and EMDP and 2.5 ng/g for methadol. The limits of quantitation (LOQ) for methadone, EDDP, EMDP were 5 and 25 ng/g for methadol. Linearity ranged from 5.0 to 500 ng/g. Following solid-phase extraction, no matrix effect was observed. This method proved to be suitable for the quantification of methadone, EDDP and EMDP and the semi-quantitation of methadol in meconium. Literature review revealed no other published LC-APCI-MS/MS method for the detection of methadone and its three main metabolites in meconium specimens.     

Endometriotic Cells Exhibit Metaplastic Change and Oxidative DNA Damage as Well as Decreased Function,Compared to Normal Endometrium

Summary A widely accepted theory of the etiology of endometriosis is that it originates from the implantation and invasion of cells from retrograde menstruation to various sites in the body particularly the pelvic peritoneal cavity. Little is known of the function of these cells in ectopic sites. Normal endometrium was compared with endometriotic tissue using an antibody to Placental Cadherin (P Cadherin), a recently studied cadherin that is implicated in metaplasia and early neoplasia and also 8-hydroxyguanine, an indicator of oxidative DNA damage. Comparisons of endometrial tissue function were made using expression of transforming growth factor β-1 (TGFβ-1) and insulin-like growth factor-I (IGF-I). There was no labelling for anti-P Cadherin or anti-8-hydroxydeoxyguanosine in normal endometrium but marked labelling for both on the apical surface of the endometriotic epithelium. Studies of markers of normal endometrial function were all de-expressed in endometriosis. This study indicates that endometriosis cells are abnormal and exhibit oxidative DNA damage, metaplasia and markedly reduced function compared to normal endometrium.     

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.     

Chromatin modification and the endothelial-specific activation of the E-selectin gene

E-selectin plays a role in the binding and extravasation of leukocytes from the bloodstream. The E-selectin gene is rapidly and transiently expressed by endothelial cells activated by inflammatory stimuli. Despite the identification of factors critical for cytokine-induced activation of the E-selectin promoter, little is known about the mechanisms that restrict the gene expression to endothelial cells. We used in vivo approaches to characterize the E-selectin promoter in primary cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells. In endothelial cells specifically, nucleosomes are remodeled after tumor necrosis factor (TNF) alpha induction. Chromatin immunoprecipitation analysis demonstrated the binding of the p65 (RelA) component of nuclear factor-kappa B (NF-kappa B) to the endogenous E-selectin promoter after TNFalpha stimulation along with IkappaB kinase alpha. Multiple coactivators, including p300, steroid receptor coactivator-1, and p300/cAMP-response element-binding protein (CREB)-binding protein (CBP)-associated factor localize differentially to the E-selectin promoter. Additionally, TNFalpha induced localized histone hyperacetylation, phosphorylation, and methylation in the E-selectin gene specifically in endothelial cells. Post-induction repression of E-selectin expression is associated with recruitment of multiple deacetylases. Collectively, these studies suggest a model for the selective induction of the E-selectin gene in which the core promoter chromatin architecture is specifically modified in endothelial cells.     

Interactions of mitochondria-targeted and untargeted ubiquinones with the mitochondrial respiratory chain and reactive oxygen species. Implications for the use of exogenous ubiquinones as therapies and experimental tools

Antioxidants, such as ubiquinones, are widely used in mitochondrial studies as both potential therapies and useful research tools. However, the effects of exogenous ubiquinones can be difficult to interpret because they can also be pro-oxidants or electron carriers that facilitate respiration. Recently we developed a mitochondria-targeted ubiquinone (MitoQ10) that accumulates within mitochondria. MitoQ10 has been used to prevent mitochondrial oxidative damage and to infer the involvement of mitochondrial reactive oxygen species in signaling pathways. However, uncertainties remain about the mitochondrial reduction of MitoQ10, its oxidation by the respiratory chain, and its pro-oxidant potential. Therefore, we compared MitoQ analogs of varying alkyl chain lengths (MitoQn, n = 3-15) with untargeted exogenous ubiquinones. We found that MitoQ10 could not restore respiration in ubiquinone-deficient mitochondria because oxidation of MitoQ analogs by complex III was minimal. Complex II and glycerol 3-phosphate dehydrogenase reduced MitoQ analogs, and the rate depended on chain length. Because of its rapid reduction and negligible oxidation, MitoQ10 is a more effective antioxidant against lipid peroxidation, peroxynitrite and superoxide. Paradoxically, exogenous ubiquinols also autoxidize to generate superoxide, but this requires their deprotonation in the aqueous phase. Consequently, in the presence of phospholipid bilayers, the rate of autoxidation is proportional to ubiquinol hydrophilicity. Superoxide production by MitoQ10 was insufficient to damage aconitase but did lead to hydrogen peroxide production and nitric oxide consumption, both of which may affect cell signaling pathways. Our results comprehensively describe the interaction of exogenous ubiquinones with mitochondria and have implications for their rational design and use as therapies and as research tools to probe mitochondrial function.     

Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone

It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 "client" protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601-607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR.hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR.hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR.hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR.hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR.hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a approximately 100-fold shift to the right in the steroid dose response for gene activation.     

Cholesterol-dependent lipid assemblies regulate the activity of the ecto-nucleotidase CD39

CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase1) is a plasma membrane ecto-enzyme that regulates purinergic receptor signaling by controlling the levels of extracellular nucleotides. In blood vessels this enzyme exhibits a thromboregulatory role through the control of platelet aggregation. CD39 is localized in caveolae, which are plasma membrane invaginations with distinct lipid composition, similar to dynamic lipid microdomains, called rafts. Cholesterol is enriched together with sphingolipids in both rafts and caveolae, as well as in other specialized domains of the membrane, and plays a key role in their function. Here, we examine the potential role of cholesterol-enriched domains in CD39 function. Using polarized Madin-Darby canine kidney (MDCK) cells and caveolin-1 gene-disrupted mice, we show that caveolae are not essential either for the enzymatic activity of CD39 or for its targeting to plasma membrane. On the other hand, flotation experiments using detergent-free or detergent-based approaches indicate that CD39 associates, at least in part, with distinct lipid assemblies. In the apical membrane of MDCK cells, which lacks caveolae, CD39 is localized in microvilli, which are also cholesterol and raft-dependent membrane domains. Interfering with cholesterol levels using drugs that either deplete or sequester membrane cholesterol results in a strong inhibition of the enzymatic and anti-platelet activity of CD39. The effects of cholesterol depletion are completely reversed by replenishment of membranes with pure cholesterol, but not by cholestenone. These data suggest a functional link between the localization of CD39 in cholesterol-rich domains of the membrane and its role in thromboregulation.     

A maturational model for the study of airway smooth muscle adaptation to mechanical oscillation

It has been shown that mechanical stretches imposed on airway smooth muscle (ASM) by deep inspiration reduce the subsequent contractile response of the ASM. This passive maneuver of lengthening and retraction of the muscle is beneficial in normal subjects to counteract bronchospasm. However, it is detrimental to hyperresponsive airways because it triggers further bronchoconstriction. Although the exact mechanisms for this contrary response by normal and hyperresponsive airways are unclear, it has been suggested that the phenomenon is related to changes in ASM adaptability to mechanical oscillation. Healthy immature airways of both human and animal exhibit hyperresponsiveness, but whether the adaptative properties of hyperresponsive airway differ from normal is still unknown. In this article, we review the phenomenon of ASM adaptation to mechanical oscillation and its relevance and implication to airway hyperresponsiveness. We demonstrate that the age-specific expression of ASM adaptation is prominent using an established maturational animal model developed in our laboratory. Our data on immature ASM showed potentiated contractile force shortly after a length oscillation compared with the maximum force generated before oscillation. Several potential mechanisms such as myogenic response, changes in actin polymerization, or changes in the quantity of the cytoskeletal regulatory proteins plectin and vimentin, which may underlie this age-specific force potentiation, are discussed. We suggest a working model of the structure of smooth muscle associated with force transmission, which may help to elucidate the mechanisms responsible for the age-specific expression of smooth muscle adaptation. It is important to study the maturational profile of ASM adaptation as it could contribute to juvenile hyperresponsiveness.